The metabolic energy required for growth, repair, and survival of eukaryotic cells is predominantly generated by mitochondria, but an unavoidable side reaction of oxidative metabolism is auto-oxidation of oxygen to superoxide. The mitochondrial theory of aging, whereby mitochondria arc both the main producers of ROS and the most vulnerable cell structures to ROS-induced damage, posits that mitochondrial DNA mutation load and/or damage to vital enzymatic activities leads to death of cells, and, ultimately, death of the organism. This theory of aging is supported by studies of aging mammals, flies, and roundworms. However, little is known about the role of mitochondrial ROS or mitochondrial damage in aging and longevity of Saccharomyces cerevisiae, an otherwise validated model for studies of organismal aging and longevity. In this proposal, we will explore specific aspects of yeast mitochondrial respiratory functions, biocnergetics, and damage in order to explore the link between mitochondrial respiration and oxidative damage during yeast aging. In addition, through cross-phyletic studies (yeast, C. elegans, and Drosophila) we will test our overall hypothesis that mitoehondrial production of ROS is a process that is amenable to regulation in vivo, such that rational interventions can be designed to modulate the links between mitoehondrial ROS, aging-associated oxidative damage, and organismal life span. This hypothesis will be tested via three specific aims. Specific Aim 1 will determine if long-term survival of yeast in stationary phase is accompanied by changes in respiratory control, state, and/or capacity. Specific Aim 2 will determine if modulation of respiration state by proteins that confer non-coupled or regulated uncoupled respiration will attenuate ROS production and aging-associated oxidative damage in yeast, C. elegans, and Drosophila. Specific Aim 3 will probe the link between mtDNA mutations and life span of yeast. These studies will contribute to our long-term goal of revealing the biochemical and physiological mechanisms of nfitoehondrial production of ROS and their effect on longevity and aging of multiple organisms. These studies also contribute to overall program goals through interactions with each of the other projects and contributions of samples to Core B for use in developing a metabolic profile that is predictive of longevity across phyla.